We continued to investigate the biological implication of membrane PS on Akt signaling that regulates cell survival. Akt activation requires phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent membrane translocation and phosphorylation, however, molecular details in Akt-membrane interaction are not clearly understood. We investigated a molecular mechanism by which constitutive membrane PS modulates dynamic PIP3-dependent Akt signaling. While PIP3 interacted primarily with the PH domain, PS interacted with both PH and regulatory (RD) domains of Akt in a concentration-dependent manner, augmenting Akt translocation, phosphorylation and activation triggered by PIP3 in living cells. We also found that either PS or PIP3 can cause conformational changes of Akt for S473 phosphorylation by mTOR for full activation, suggesting a salvaging role of PS when PIP3 production is limited. During this period, we produced further evidence that PS plays a crucial role in Akt translocation and subsequent conformational changes for phosphorylation and activation. We have previously demonstrated that the increase of membrane PS by DHA supplementation in Neuro 2A cells promoted Akt membrane translocation and phosphorylation. To exclude possible off-target effect of the DHA supplementation, we evaluated Akt activation after altering the plasma membrane PS in living cells using two DHA-independent approaches, namely employing CHO mutant cells lacking PS synthase-1 (PSA-3) where the PS content can be lowered and overexpressing PSS1 mutant R95K which is known to be resistant to the product inhibition, thus increasing the PS content. In contrast to DHA supplementation, the PSA-3 mutant cells contained a significantly lower level of plasma membrane PS, and showed noticeably inhibited IGF-triggered membrane translocation and phosphorylation of Akt in comparison to the wild type CHO cells. The observed impairment of Akt activation was not due to inhibited PI3 kinase as the PI3 kinase activity was found to be similar between the PSA-3 cells and WT. In another model where the plasma membrane PS content was raised by expressing the R95K mutant of PSS1, Akt phosphorylation was increased, while the PI3 kinase activity remained unchanged. The results obtained from these independent models consistently indicated that membrane translocation and phosphorylation of Akt are promoted in living cells with a higher PS content, strongly suggesting general involvement of plasma membrane PS in the activation of the survival Akt protein. Using biomolecular interaction analysis based on surface plasmon resonance (SPR), we have further demonstrated the PS-Akt interaction is driven by electrostatic force. Nevertheless, other negative charged membrane components including PI, PIP, and PIP2 did not interact with Akt, indicating specifc electrostatic interaction between Akt and membrane PS. During this period, we identified PS-interacting residues in the regulatory domain (RD) by SPR and mutation of basic residues in the RD, and confirmed R15 and K20 in the PH domain as PS binding residues. We demonstrated that Akt translocation and activation in living cells was impaired by disrupting Akt-PS interaction by mutating PS-binding residues of Akt, indicating that the observed Akt-membrane PS interaction is crucial and physiologically relevant for Akt activation. The novel molecular interaction mechanism in Akt signaling revealed in our study during this review period may provide an insight for potential new targets for controlling cell survival. We also established a method for profiling the downstream signaling events of Akt activation by searching for Akt-binding proteins and/or phosphoproteins. Phosphoproteins or Akt-interacting proteins immuno-purified using Akt antibody (or Flag antibody for expressed Flag-Akt), were digested using trypsin, labeled with 18O water or isotope tags and analyzed by nano-LC/ESI-MS/MS. Using this approach, several potential Akt interacting proteins were identified during the course of IGF stimulation. Phosphopeptides were further enriched using titanium oxide prior to mass spectrometric analysis. This approach enabled us to unveil minor phosphopeptides generated during stimulation of the cells. This approach is now being applied to the investigations of the Akt interacting phosphoproteins during stimulation with an aim to investigate the effect of DHA supplementation and/or ethanol on Akt interaction profile and phosphorylation. As the PS accumulation is an important aspect of DHA- or ethanol-mediated effects on Akt signaling, we continued to characterize the neuronal PS accumulation. During this period, we were able to clone microsomal phosphatidylserine synthase 2 (PSS2) from mouse brain, one of the enzymes responsible for PS synthesis, which was subsequently expressed in neuronal cells and successfully purified with activity preserved. While structural and biochemical characterization of the purified PSS1 at the molecular level is in progress, we also developed a method to evaluate the PS remodeling processes in living cells by monitoring stable isotope labeled serine incorporation into specific phospholipid pools using mass spectrometry. We will subsequently apply this method to elucidate the mechanism for neuronal-specific PS accumulation promoted by DHA supplementation. To identify lipid mediators derived from DHA, we have established an HPLC/ESI-MS/MS method during this period using uniformly 13C -labeled DHA (13C-DHA) together with non-labeled DHA. Based on the unique isotope profile of 13C-DHA-derived metabolites together with the predicted mass differences from the corresponding 12C-DHA-derived metabolites, we have identified the formation of DHA metabolites in developing hippocampi including N-docosahexaenoylethanolamide (DEA) as well as monohydroxy and dihydroxy-DHA. The production of N-docosahexaenoylphosphatidylethanolamine (NDPE), a structural analogue of N-arachidonyphosphatidylethanolamine (NAPE), was also detected, suggesting that biochemical mechanism of DEA production may be similar to that of the well-known endocannabinoid N-arachidonylethanolamine (AEA). The putative DEA precursor NDPE was identified as diverse molecular species of plasmalogens (p16:0/22:6 and p16:0/20:4) and diacyl species (16:0/22:6, 16:0/18:1, 18:0/20:4 and 18:0/18:1). The stable isotope-assisted mass spectrometric approach may be easily extended to survey the formation of a broader range of bioactive metabolites with the help of an automatic peak finding algorithm. We also found during this period that DEA production is directly linked to the DHA level and is most active during development, suggesting that DHA metabolism to DEA is an important mechanism for neurodevelopment and function.